(1) We refined data from the last year on detergent-resistant membrane and lipid profiles of human erythrocytes containing hemoglobin C, a mutant hemoglobin known for to protect children against severe forms of falciparum malaria in West Africa. The manuscript has been published for the publication in PLoS ONE. (2) We continued to expand our research project on intracellular trafficking of P. falciprum-derived proteins using tetracysteine-tag for fluorescence imaging. Using genetically engineered knob-asscociated histidine rich protein (KAHRP) tagged with tetracysteine sequence, we successfully established the labeling and observation technology for dynamic intracellular protein trafficking, which is expected to overcome shortcomings of current GFP technology and enables us for pulse-chase labeling. The manuscript based on this work is under preparation. (3) We started a new project using fluorescence lifetime microscopy (FLIM) to study molecular and membrane dynamics of P. falciparum-infected human erythrocytes. Using an environment-sensitive fluorophore, Di-4 ANEPPDHQ and time-correlated fluorescence lifetime aquisition, we found distinguished characteristics of parasitophorous vacuole and Maurer's clefts, two membranes developed by intracellular stages of P. falciparum. and critical for parasite-derived protein traffickings. The data indicated that unique properties of these de novo membrane systems may provide a hint for a complex protein trafficking in P. falciparum-infected erythroyctes. (4) We continued developping Total Internal Reflection Microscopy (TIRFM), and combined with spinning-disk confocal imaging. These technologies are used for Single Molecule Tracking (SPT) to study dynamic molecular diffusions and interactions of malaria parasite-derived proteins on human erythrocyte membrane. This project is augmented by cutting-edge imaging technologies, such as Quantum Dots-based labelling of target molecules. A new substrate was also developed for minimized thermal fluctuations but retaining normal biconcave shape of erythrocytes. (1) We made an effort to improve data quality in submitted manuscript in the first half of this fiscal year. This work was successfully published in Jun 6 edition of PLoS ONE, 4(6): e5828. (2) Study of the trafficking of knob-forming parasite-derived proteins in human erythrocyte is important for identifying elements in the pathogenesis of the disease. Common method for protein labeling to track proteins of interest is expressions of GFP-fusion proteins. However, the exact mechanism of the transport may be obscured by the large size of GFP moieties and tendency of GFP to aggregate. For more precise measurement, we developed a new protein labeling strategy, employing recombinant proteins engineered to express tetracysteine (TC) tags, which can be further labeled with red (ReAsH) and green (FlAsH) biarsenical fluorophores. In this project, we successfully engineered KAHRP-GFP-TC and KAHRP-TC constructs. ReAsH-TC labeling of KAHRP-GFP-TC fusion proteins confirmed expression of TC tag and a comparable fluorescence pattern of protein distribution as GFP fluorescence, showing the capability of TC tag to be used for study of protein trafficking in a live condition. In addition, existing KAHRP-TC proteins at one time point were presented with one label while the newly synthesised proteins after 5 hours were chased with another label. This pulse-chase labeling technique enabled us to identify transitional localizations of target proteins synthesized at different times during intraerythrocytic stages of the parasite. These results show that the tetracystein technology can provide a new aspects of protein trafficking mechanism which is not possible with the common GFP-labelling. (3) Intracellular stages of P. falciparum induce unique host cell modifications. Parasites are surrounded by erythrocyte membrane-derived parasitophorous vacuole (PVM) which continuously increase in its size as parasites mature. In addition, new membranes emerge from PVM and form eccentric membrane structures in erythrocyte cytoplasm, called Mauere's cleft. This structure seems to play crucial roles in trafficking and delivery processes for parasite-derived proteins. However, the source of lipid molecules to support the size increase in the new membrane structure is unknown. A key to this question is cholesterol content as parasite has no cholesterol synthesis mechanism. If parasites supplement their own lipids to these new membrane systems, cholesterol content should have different level as compare to host erythrocyte membrane. In addition, cholesterol is one of the most important molecules for membrane biophysical properties. To study membrane properties of PVM and Mauere's cleft, we applied Fluorescence Lifetime Microscopy (FLIM) and fluorophores sensitive to cholesterol-rich membrane domains. We successfully obtained a clear differences in FLIM data between erythrocyte membrane, Maurer's cleft, PVM, and parasite membranes, showing PVM and Maurer's cleft have unique properties which are mixtures of erythrocytes membrane and lipids from parasites. This result may suggest that a portion of Maurer's cleft membrane comes from PVM containing parasite-derived lipids. The manuscript for these data is now under preparation. (4) In this fiscal year, we continued developing single-molecule tracking technology for P. falciparum-infected erythrocytes to monitor intermolecular interactions in real-time on live cells. This is a challenging project and never done in other laboratories. We have completed implementations of both Total Internal Reflection Fluorescence Microscopy (TIRFM) combined with spinning disk confocal. These technolgies will increase signal-to-noise ratio of acquired images by excluding undesired backgrounds fluorescence from out-of-focus planes. As preliminary experiments, we labeled CD47 with quantudm dots and tracked single CD47 molecules in live cells. This experiment showed that the diffusion coefficient of CD47 has two subpopulations: immobile and mobile pools. We found that P. falciparum infection reduced significantly the mobile pool of CD47, showing that P. falciparum infection dramatically modifies the erythrocyte membrane protein mobility. However, thermal fluctuations of erythrocyte membrane reduce accuracy of diffusion data. To overcome this problem, we also developed a customized substrate surface condition using regular poly-L-lysine combined with gold nanoparticles. This combination retains necessary cell absorption force, while reducing excess force by non-polar gold nanoparticles. We successfully found an optimal condition for attached live erythrocytes with normal biconcave shape but minimal thermal fluctuations. Collaborative Projects (National Institute of Standards and Technology) In this fiscal year, we collaborated with National Institute of Standards and Technology to modify commercial based microscope to be capable of TIRFM. This collaboration also developed a custom made image analysis routines allowing us to perform simultaneous multi-molecule tracking at sing-molecule resolution. We also developed a new substrate for single particle tracking described in (4). (Tokyo Women's Medical School) We start observing PVM membrane structure by electron microscope to study parasite-derived translocon protein structures for protein trafficking across the PVM. Although the translocon protein complex has been suggested biochemically, structure and distribution of the complex remain to be studied. We will employ 3D cryo-electron mircosocpy to directly visualize the structure of these proteins. We have obtained preliminary data showing large channel-like structure distributed on PVM membrane.